Prior studies implicate the role of the reactive retinal aldehyde, all-trans-retinal (atRAL) in degenerative retinal diseases such as Stargardt's disease and age-related macular degeneration and contributions of inflammatory responses to those diseases. Although continuous generation of atRAL by the visual (retinoid) cycle is required to maintain vision, chronic exposure to atRAL may serve as an endogenous stress inducer that can cause low-grade chronic inflammation. Two characteristic features of low-grade chronic inflammation in the retina are complement deposition at Bruch's membrane and invasive immune cells into the subretinal space. Our previous studies provided the following notable findings: (1) Rdh8-/-Abca4-/- mice with delayed atRAL clearance from the retina that develop cone-rod dystrophy also display complement deposition at Bruch's membrane. (2) Subretinal accumulation of microglia/macrophages in Rdh8-/-Abca4-/- mice. (3) Tlr3-/- Rdh8-/-Abca4-/- mice displayed milder retinal degeneration with less inflammatory changes compared to Rdh8-/- Abca4-/- mice. These observations strongly indicate that retinal inflammation is caused by the biological responses to atRAL, and this inflammation greatly contributes to age-related disorders of the retina. We propose three specific aims to examine the role of atRAL in retinal inflammation that may contribute to retinal tissue damages. In Aim 1, the role of atRAL for recruitment of immune cells to the subretinal space will be examined. Retinal microglial behavior will be investigated in Rdh8-/-Abca4-/- mice after light which causes accumulation of atRAL using in vivo scanning laser ophthalmoscopic and two-photon microscopic imaging and immunohistochemical analysis. Changes in chemokine/cytokine production in the retina of Rdh8-/-Abca4-/- mice will be examined with RNA analyses. Aim 2 will test the hypothesis that CCL3 is a major chemokine that mediates recruitment of inflammatory cells to the subretinal space in retinal degeneration caused by atRAL accumulation, because our preliminary study show ~80-fold increase of CCL3 production along with increased numbers of immune cells in the subretinal space of Rdh8-/-Abca4-/- mice. Effects of CCL3 will be investigated in degenerative phenotypes of Rdh8-/-Abca4-/- mice by creating Ccl3-/-Rdh8-/-Abca4-/- mice, and by subretinal CCL3 injection to WT mice. TLR3-medicated CCL3 production will also be examined using Tlr3-/-Rdh8-/-Abca4-/- mice that showed milder retinal degeneration with less inflammatory changes. In Aim 3, we will test the hypothesis that invasive immune cells contribute to retinal tissue damage by employing macrophage-depletion in mice with genetic and pharmacologic approaches and drug treatments with microglial inhibitors to Rdh8-/- Abca4-/- mice, and by in vitro experiments using co-culture of primary RPE and retinal microglial cells. Experimental results of these interrelated aims outlined in this application will determine the rol of atRAL in retinal inflammation and degeneration, and also will aid in the development of a new therapeutic concept whether atRAL is an appropriate therapeutic target for age-related retinal disorders. PUBLIC HEALTH RELEVANCE: The number of persons who are legally blind in the USA exceeds 1.3 million with 8-10 million aging persons who are afflicted with retinal degenerative diseases. Utilizing a mechanistically relevant model of Stargardt's macular degeneration with similar phenotypes observed in age-related macular degeneration, we have demonstrated that all-trans-retinal is one of the strongest candidate targets for the treatment of these grievous diseases. The goal of this proposed research is to characterize the detrimental effects of all-trans-retinal in retinal degeneration and to develop improved therapies by targeting this molecule.